融合蛋白ELP30-Trx的表达及PEG8000对其相变温度的影响  

Expression of Fusion Protein ELP30-Trx and Influence of PEG to ELP30-Trx Inverse Temperature Transition

作　　者：张立超[1] 葛高顺[1] 胡学军[1] 

机构地区：[1]大连大学医学院医学研究中心,辽宁大连116622

出　　处：《生物技术》2013年第5期36-39,共4页Biotechnology

基　　金：国家自然基金项目("单链抗体在大肠杆菌体内定点糖基化及其通过糖链以共价键定向固定的研究";No.31070822/C080602);辽宁省高等学校科研项目计划项目("糖基化多肽聚合物的生物合成及其抑制毒素的活性研究";No.L2010015);辽宁省大学生创新创业训练计划项目(201211258012)资助~~

摘　　要：目的:研究不同浓度的PEG8000对融合蛋白ELP30-Trx相变温度的影响。方法:将硫氧还蛋白(Thioredoxin,Trx)基因克隆到pET28-ELP30-Trx表达载体中,经IPTG诱导在宿主菌E.coli BLR(DE3)中表达ELP30-Trx,通过可逆相变循环(Inverse transition cycling,ITC)纯化融合蛋白。测量相变温度(T t),并检测不同浓度的PEG8000对ELP30-Trx T t的影响。结果:经酶切及测序鉴定证实成功构建了ELP30-Trx表达载体,经Westen-Blot检测表明成功表达ELP30-Trx融合蛋白。经ITC纯化后的ELP30-Trx纯度可达95%以上。5%、10%、15%、20%的PEG8000(W/V)使终浓度为25μmol/L ELP30-Trx的T t从37.9℃分别降低至34.6℃、28.5℃、14.0℃、6.0℃。结论:成功构建、表达融合蛋白ELP30-Trx,PEG8000能显著降低ELP30-Trx相变温度。通过对类弹性蛋白的研究,为类弹性蛋白的扩大应用提供理论依据。Objective： To study the influence of different concentrations of PEG to fusion protein （ ELP30 - Trx） inverse temperature transi-tion（ Tt ）. Method： Thioredoxin gene was inserted into pET28 which contain ELP30 to obtain the expression plasmid pET28 - ELP30 - Trx,The fusion protein was expressed in E. coli BLR（ DE3 ） induced by IPTG, and was confirmed via SDS - PAGE and Westen - blo-ting. The fusion protein was purified via ITC （Inverse transition cycling）, then the Tt was tested under the condition of different concentra-tions of PEG8000. Result： The pET28 - ELP30 - Trx was constructed successfully. The ELP30 - Trx fusion protein was expresssed and purified via ITC. The purity of fusion protein was up to 95%. The 25 μmol/L fusion protein＇ s T,s were reduced respectively to 34. 6℃, 28.5 ℃ ,14. 0 ℃ ,6. 0 ℃ by 5% ,10% ,15% ,20% （W/V） PEG8000 from 37. 9℃. Conclusion： The PEG8000 can reduce the ELP30 fusion protein＇ s Tt ,which laid the foundation for the application of ELPs.

关 键 词：类弹性蛋白 纯化标签 PEG8000 融合蛋白 硫氧还蛋白 

分 类 号：Q819[生物学—生物工程]